Seed cryopreservation and longevity of two Salix hybrids

The effects of desiccation and storage temperature on the viability and longevity of willow seeds was investigated using two hybrids, Salix rehderiana x (Salix x capreola) [cross 458] and Salix x sericans x Salix viminalis [cross 512]. Freshly harvested seed of both crosses survived silica gel drying down to c. 3 to 5% moisture content. Hybrid 458 seed stored in liquid nitrogen (-196C) for 3 d retained viability when equilibrated to < or = 45% RH (pre-storage), showed slightly reduced survival at 65% RH and exhibited no survival at > or =82% RH. The level of survival after 68 d for seeds pre-equilibrated to either c. 10 or 65% (5 or 10% moisture) and stored at four temperatures was -196C > -20C > 2C > 16C. At all temperatures, drier seed stored better than wetter seed. For hybrid 512, seed longevity at 20C > 40C > 60C, and a 10% fall in pre-storage seed RH resulted in a c. 2-fold increase in longevity at each storage temperature. The response of hybrid willow seeds to desiccation and cooling raises possibilities for the long-term seed conservation of Salix species by cryopreservation.     

Narrowing oF the critical hydration window for cryopreservation of Salix caprea seeds following ageing and a reduction in vigour

We investigated the effects of desiccation, rehydration and cryopreservation on the viability of seeds of a wild mountain species and seven clones of Salix caprea L. Seeds responded differently to all treatments depending on clone, seed initial moisture content (MC) and seed vigour. Fresh seeds of two randomly selected clones tolerated desiccation to MC 8.5-9.6 % FW (0.09-0.11 g water per g dry mass. g/gdw) without any noticeable loss in viability and were successfully cryopreserved at MCs ranging from 8.5 to 23.4 % (0.09-0.30 g/gdw). Storage at 5 degree C for approximately 10 weeks significantly reduced the viability of seed lots of a wild species and of three S. caprea clones, whilst viability of seeds of four other clones remained unaffected. Since all clones tested were genetically derived from one tree, this variation is unlikely to be of maternal origin. Most probably paternal x environmental factors have influenced seed behavior during desiccation and storage. As viability decreased due to partial ageing, seeds became more susceptible to desiccation stress. When seeds of three clones were cryopreserved, the hydration window for survival was wider for highly vigorous seeds (c. 0.05-0.28 g/gdw) than for seeds with intermediate vigour (c. 0.10-0.24 g/gdw) and low vigour (c. 0.20-0.37 g/gdw). Rehydration to MC above 0.15 g/gdw improved germination of low vigour seeds, both in controls and after cryopreservation. In contrast, cryopreservation of high vigour seeds rehydrated to MCs above 0.11 g/gdw resulted in a sharp decrease in normal seedling production. Whilst no effect of cryogenic temperature on germination and normal seedling production was observed when seeds of seven clones were cryopreserved within their hydration windows, the results indicate the need to account for seed lot vigour when designing cryopreservation protocols.     

High viability of dormant Malus buds after 10 years of storage in liquid nitrogen vapour

Three hundred and sixty two Malus accessions from the Canadian Clonal Genebank of Plant Gene Resources of Canada were cryopreserved as dormant buds at the USDA-ARS National Center for Genetic Resources Preservation in 1996. According to grafting data collected on 165 of these accessions in 1999, 80 percent of the accessions had at least 40 percent viability. A subsample of these accessions was processed for cryopreservation by either adjusting the moisture content of the budwood sections containing dormant buds to 32 or 37 percent moisture (fresh weight basis) or by not drying the budwood sections (46 percent moisture fresh weight basis) prior to cooling. Budwood sections were then slow-cooled at 1 degree C per hour to -3 degree C, held for 24 h at -30 degree C and then rapidly transferred to the vapour phase of liquid nitrogen. Cryopreserved buds from 13 accessions that were dried using the various techniques were warmed and grafted in both 1999 and 2006 to determine viability. Overall, bud viability was high at both storage times. At the 10 year time point, some accessions had higher bud growth when they were desiccated prior to slow-cooling when compared to those that were not.     

Low temperature survival of slender naiad (Najas flexilis) seeds

The response to drying and storage at -20 degrees C or in liquid nitrogen was studied in seeds of the freshwater aquatic plant Najas flexilis. The seeds of this species show some desiccation sensitivity, although post-harvest storage in water at 16 degrees C resulted in improvements in desiccation tolerance. There was 63% germination of seeds dried to 9.5% moisture content (30% RH) following this maturation period. Optimum moisture contents for seeds stored at -20 degrees C for 3 months and in liquid nitrogen for 1 week were ~11% and ~15%, respectively.     

Cryopreservation of cryosensitive basidiomycete cultures by application and modification of perlite protocol

Homolka's perlite protocol (HPP) for cryopreservation of fungal cultures was evaluated in 12 strains (7 species) of cryosensitive basidiomycete cultures maintained in NBRC culture collection by investigating viability, time to recover, and basic morphological study after freezing and storing at -80 degree C for 6 months. The viability of the fungal strains was 60 percent in Phallus hadriani and 100 percent in remaining 11 strains, indicating the efficacy of HPP method for cryopreservation of some cryosensitive basidiomycetes. The HPP method was modified by changing the addition of cryoprotectant (glycerol) from prior precultivation to post precultivation, limiting the cryoprotectant exposure time to 48 hours, and increasing the glycerol concentration from 5 percent to 12 percent. The viability of P. hadriani strain increased from 60 percent to 100 percent with the modified perlite protocol after storage at -80 degree C for 6 months.     

Pollen from Glycine species survive cryogenic exposure

Pollen of 12 genotypes of the annual soybean and its wild perennial relatives were stored without pre-desiccation at low temperatures (-20 C and -196 C) and tested for their viability in vitro. The influence of cryopreserved pollen on pod set and seed production was also investigated. Cryopreserved pollen of all the genotypes showed germination in vitro. Pollen of annual soybean stored at -20 C retained their viability for 4 months, however, pollen of its wild perennial relatives at same storage conditions failed to germinate in vitro. Flowers pollinated with cryopreserved pollen had similar pod set and number of seeds/pod as those pollinated with fresh pollen. Results of this study suggest that cryopreservation of pollen can be used successfully for soybean breeding, and also offers the possibility of conserving the haploid gene pool of soybean and wild perennial species in a cryobank facility.     

The effects of desiccation and exposure to cryogenic temperatures on embryonic axes of Landolphia kirkii

The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates on survival, assessed by the ability for both root and shoot development, of embryonic axes excised with a small portion of each cotyledon, from mature, recalcitrant seeds of Landolphia kirkii. All axes withstood rapid (flash) drying to a water content of c. 0.28 g water per g dry mass; however, the use of chemical cryoprotectants before flash drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g water per g dry mass, reducing survival to 10% and 0% after exposure to -196 degree C and -210 degree C, respectively. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent rewarming and rehydration. Cooling at 1 degree C per min facilitated survival of 70% of axes with attached cotyledonary segments at 0.28 g water per g dry mass after exposure to -70 degree C, with 45% viability retention when further cooled at 15 degree C per min to -180 degree C. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached.     

Cryopreservation of 'Nabali' olive (Olea europea l.) somatic embryos by encapsulation-dehydration and encapsulation-vitrification

Olive (Olea europea L.) somatic embryos were successfully cryopreserved using encapsulation-dehydration and encapsulation-vitrification. In the encapsulation-dehydration procedure, a maximum of 48% embryo survival was obtained when bead moisture content was decreased to 21.1% after 4 h dehydration. Preculture of embryos for 4 d in medium containing 0.75 to 1.25 M sucrose produced higher (40 to 34 %, respectively) regrowth after cryopreservation using encapsulation-dehydration procedure. Dehydration of beads for 3 h in PVS2 ensured higher survival (64%) of encapsulated-vitrified and cryopreserved (EVN) somatic embryos. Thermal treatment of embryogenic callus for 1 d at 30 degree C was very effective to increase survival of encapsulated-dehydrated and cryopreserved (EDN) (58%) and EVN (68%) embryos. Plantlets produced from control and cryopreserved embryos were phenotypically similar.     

Cryopreservation of Citrus madurensis embryonic axes encapsulation-dehydration

In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation-dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content.     

Cryopreservation of peach palm zygotic embryos

Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3 percent sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83 percent and 87 percent to 18 percent and 20 percent for pre-treated or non-pretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20 percent water content, followed by rapid freezing in liquid nitrogen (-196 degree C) and rapid thawing at 45 degree C. In these conditions 29 percent of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41+/-9 percent and 88+/-4 percent survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively.     

Cryopreservation of in vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration

In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydration with PVS2 for 90 min at 0 degree C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40 degrees C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40 degree C. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.     

Thermal analysis of the plant encapsulation-dehydration cryopreservation protocol using silica gel as the desiccant

The encapsulation-dehydration cryopreservation protocol is critically dependent upon the evaporative desiccation step, which must optimise survival with the retention of glass stability on sample cooling and rewarming. Desiccation is usually achieved evaporatively by drying in a sterile airflow. However, chemical desiccation using silica gel has advantages for laboratories that do not have environmental control and/or which are exposed to high relative humidities and risks of microbial contamination. This study characterised thermal profiles of silica gel-desiccated encapsulated shoot-tips of two Ribes species using Differential Scanning Calorimetry. For both species silica gel-desiccation at 16 degrees C for 5 h decreased bead water content from ca. 75 to 28% fresh weight (3.8 to 0.4 g x g(-1) dry weight); further desiccation (for 6 and 7 h) reduced the bead water content to 21% (0.3 g x g(-1) dry weight). These changes in water status altered the thermal properties of beads for both species. After 7 h desiccation over silica gel stable glass transitions were observed on both cooling and rewarming of beads containing meristems. Tg mid-point temperatures ranged from -78 to -51 degrees C (cooling) and from -88 to -54 degrees C (warming) [at cooling and warming rates of 10 and 5 degrees C min(-1), respectively] after 5 to 7 h silica gel-desiccation. Post-cryopreservation viability of both species was ca. 63%. Thermal analysis studies revealed that an encapsulation/dehydration protocol using silica gel as a desiccant should comprise a minimum 5 h drying (at 16 degrees C). This reduces bead moisture content to a critical point (ca. 0.4 g x g(-1) dry weight) at which stable glasses are formed on cooling and rewarming. It is concluded that silica gel has advantages for use as a desiccant for alginate-encapsulated plant meristems by promoting stable vitrification and is useful in laboratories and/or geographical locations where environmental conditions are not under stringent control.     

The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance

The effects of fruit maturity, at the time of natural dispersal, on subsequent desiccation tolerance and sub-zero storage was investigated in three lots of Acer pseudoplatanus (sycamore) collected from northern to southern Europe. Fruits from the native plant distribution range in Italy had significantly higher desiccation tolerance (0.16 g water per g DW) than those from England (0.30) and Norway (0.50), confirming that the maximum potential desiccation tolerance in sycamore exceeds that of the recalcitrant type. In contrast, the unfrozen water content varied only slightly between seedlots, but systematically reduced with development (0.35 to 0.27 g water per g DW). Maximum survival (60 percent fruit germination) of seven days sub-zero temperature storage coincided with drying the Italian fruit lot to c. 0.2 g water per g DW followed by holding at -20 degree C, above the onset temperature for freezing, or at -196 degree C (liquid nitrogen). Fruit survival was much lower in the Italian fruits when held at this water content and -70 degree C, and in all other combinations of water content, temperature and fruit lot provenance. As the risk of nucleation in partially dried fruits held at -20 degree C is high, we recommend sycamore fruits are cryopreserved for long-term conservation.     

Beneficial effect of post -thawing osmoconditioning on the recovery of cryopreserved coffee (Coffea arabica L.) seeds

Osmoconditioning-controlled rehydration of seeds in a solution with low osmotic potential -has been shown to reinvigorate aged seeds. The present work aimed at investigating the effect of osmoconditioning on the germination of cryopreserved seeds of Coffea arabica, whose viability and vigour are drastically affected by cryopreservation. For cryopreservation, seeds were desiccated to 0.21 g H2O/g dw, cooled at 1 degree C/min to -50 degree C, then immersed rapidly in liquid nitrogen. After rapid rewarming, seeds were osmoconditioned for 1 to 6 weeks using solutions with osmotic potentials between -1 and -4 MPa. The time to produce half of the final percentage of normal seedlings, T50, was about three fold lower with osmoconditioned seeds than with non-osmoconditioned seeds (12-14 d vs 36 d). Moreover, after a 6-week osmoconditioning treatment with solutions with osmotic potential of -1 and -1.25 MPa, the percentage of seedlings recovered from cryopreserved seeds was 64-74%, against 13-16% only for cryopreserved seeds which were not osmoconditioned.     

Cryopreservation of seeds of a Thai orchid (Doritis pulcherrima lindl. ) by vitrification

Seeds from selfing of a Thai orchid (Doritis pulcherrima Lindl.) were successfully cryopreserved in liquid nitrogen (LN) using the vitrification method. Seeds from 3-month-old pods were sufficiently dehydrated in 2 ml cryotubes filled with highly concentrated vitrification solution (PVS2) at 25 +/- 2 degree C for 50 min. The seeds were then rapidly plunged into LN. After rapid warming, the PVS2 solution was replaced with 0.5 ml of 1.2M sucrose in modified Vacin and Went (1949) (VW) solution and kept at 25 +/- 2 degree C for 20 min prior to transfer on VW agar medium. About 62% of cryopreserved seeds treated with PVS2 solution were able to develop into normal seedlings while without that treatment there was no survival. This vitrification protocol appears to be a promising technique for the cryopreservation of some Thai orchid germplasm     

Development of a shoot-tip vitrification protocol and comparison with encapsulation-based procedures for plum (Prunus domestica L.) cryopreservation

Cryopreservation of plum (Prunus domestica L.), cv Regina Claudia, was obtained by a vitrification/one-step cooling procedure of shoot tips from cold-hardened in vitro-grown plants. Best survival (57%) was obtained when the shoot tips were precultured at 4 degree C for 2 days on 0.09 M sucrose-containing Quoirin and Le Poivre medium, loaded for 30 min with a cryoprotectant (2 M glycerol and 0.4 M sucrose), incubated with the PVS2 solution at 0 C for 90 min, and directly plunged into liquid nitrogen. After re-warming in a waterbath at 40 degree C, the shoot tips were washed in a 1.2 M-sucrose MS solution for 20 min and finally plated on a regrowth medium. In comparison with the one-step cooling procedure, both the slow cooling (-0.5 degree C/min up to -45 degree C), and the two-step cooling (-160 degree C for 25 min, then -196 degree C) gave lower percentages of shoot-tip survival. Among the other cryogenic procedures tested, the performance of the encapsulation-vitrification method was similar to the vitrification protocol in terms of shoot-tip regrowth (47.5%), while encapsulation-dehydration was unsatisfactory.     

Cryopreservation of Vanda coerulea protocorms by encapsulation-dehydration

Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2 percent Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 +/- 3 degree C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 +/- 3 degree C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40 degree C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40 percent was observed with cryopreserved beads with 35 percent water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.     

高光谱技术无损检测单粒小麦种子生活力的特征波段筛选方法研究

种子活力是种子质量的一项重要指标,高活力的种子具有较强的抗逆性、生长优势及生产潜力。而种子活力在种子生理成熟时最高,随后随着贮藏时间的延长而发生着自然不可逆的降低。因此,在播种前及时、准确地对种子活力进行检测和筛选具有重要的实践意义。针对传统种子活力检测方法存在的操作过程复杂繁琐、耗时长、重复性差且对种子有破坏性等缺点,研究尝试利用高光谱成像技术建立单粒小麦种子生活力快速、无损、精确的检测方法。以高温高湿老化后的190粒小麦种子（发芽128粒,不发芽62粒）作为研究样本,先利用可见-近红外(Vis-NIR)高光谱成像系统采集样本种子的光谱图像和进行标准发芽试验,并确保光谱采集试验和标准发芽试验的小麦种子一一对应。随后提取种子光谱图像的感兴趣区域并对其光谱数据进行平均和特征分析。分别采用一阶导数（FD）、均值中心化（MC）、正交信号校正(OSC)和多元散射校正（MSC）对原始光谱数据进行预处理,结合偏最小二乘辨别分析（PLS-DA）建立全波段PLS-DA模型,比较分析,并筛选出最适预处理方法。分别利用无信息变量消除算法（UVE）、竞争性自适应重加权算法(CARS)、连续投影算法(SPA)及耦合不同变量筛选方法对特征波段进行筛选提取,再分别基于所提取出的特征波段建立PLS-DA定性判别模型,对比分析,最终确立提取与单粒小麦种子生活力相关性最高的高光谱特征波段方法体系。结果表明：不同光谱预处理建立的模型其表现有所差异,在MC,FD,OSC和MSC中,采用MC对原始高光谱数据进行预处理,建立的全波段MC-PLS-DA判别模型,其校正集和预测集对小麦种子生活力的整体鉴别正确率分别为82.5%和83.0%,优于原始及其他预处理后建立的全波段PLS-DA判别模型,其校正集和预测集对小麦种子活种子鉴别正确率分别为94.8%和90.6%。进一步对比3种单特征波段提取方法及其耦合分析建模中,发现3种变量筛选方法耦合（UVE-CARS-SPA）的方式能够将光谱全波段的688个变量压缩至8个变量（473,492,811,829,875,880,947和969 nm）,利用所筛选出的8个变量建立的MC-UVE-CARS-SPA-PLS-DA模型获得了最优秀的鉴别效果,其校正集和预测集对小麦种子生活力的整体鉴别正确率分别为86.7%和85.1%,较全波段模型（MC-Full-PLS-DA）分别提升了4.2%和2.1%,活种子的鉴别正确率分别为93.8%和84.4%,经过此优秀模型筛选后,种子批最终发芽率可达到93.1%。实验结果表明,基于高光谱成像技术结合UVE-CARS-SPA-PLS-DA模型能够实现对单粒小麦种子生活力的定性判别。研究工作为小麦种子活力的快速、精确且无损的检测提供理论支持。     

蔬菜种子的干燥动力学及其活性

本文研究了初含水率、干燥周期、料层厚度相同时,不同供热方式（热风、热风与辐射、热风与辐射并加湿）的白菜种子在固定床的干燥动力学及其活性,与传统的只热风供热相比,当干球温度相同时辐射加热风干燥的种子终含水率比低１３．８％、发芽率比高０．３％,而辐射加热风并加湿干燥其终含水率比低５．８％,发芽率比高０．８％。为确保种子活力,建立了褚－杨白菜种子临界温度Ｔｃｖ方程,并已验证其正确性．该方程为蔬菜种子干燥提供了理论基础并有重要的工程意义。     

Cryopreservation of somatic embryos of paradise tree (Melia azedarach L.)

In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25 degrees C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation techniques tested for freezing paradise tree somatic embryos, namely desiccation, encapsulation-dehydration and pregrowth-dehydration, only encapsulation-dehydration and pregrowth-dehydration led to successful results. The optimal protocol was the following: i) somatic embryos (encapsulated or not) pretreated in liquid Murashige & Skoog medium with daily increasing sucrose concentration (0.5 M/0.75 M/1.0 M); ii) dehydrated with silica gel to 21 - 26% moisture content (fresh weight basis), for encapsulation-dehydration, or to 19% moisture content, for pregrowth-dehydration; iii) frozen at 1 degree C/min from 20 degrees C to -30 degrees C with a programmable freezing apparatus; iv) rapid immersion in liquid nitrogen. The highest recovery achieved was 36% with encapsulation-dehydration and 30% with pregrowth-dehydration. Regrowth of frozen embryos was direct in most cases, as secondary embryogenesis originating from the root pole was observed on only around 10% of cryopreserved somatic embryos. Plants recovered from cryopreserved embryos presented the same phenotypic traits as non-frozen control plants.